The use of new, deep sequencing technologies has greatly accelerated microRNA discovery. We have applied this approach to the identification of chicken microRNAs and to the comparison of microRNAs in ...chicken embryo fibroblasts (CEF) infected with Marek's disease virus (MDV) to those present in uninfected CEF.
We obtained 125,463 high quality reads that showed an exact match to the chicken genome. The majority of the reads corresponded to previously annotated chicken microRNAs; however, the sequences of many potential novel microsRNAs were obtained. A comparison of the reads obtained in MDV-infected and uninfected CEF indicates that infection does not significantly perturb the expression profile of microRNAs. Frequently sequenced microRNAs include miR-221/222, which are thought to play a role in growth and proliferation. A number of microRNAs (e.g., let-7, miR-199a-1, 26a) are expressed at lower levels in MDV-induced tumors, highlighting the potential importance of this class of molecules in tumorigenesis.
Deep sequencing technology is highly suited for small RNA discovery. This approach is independent of comparative sequence analysis, which has been the primary method used to identify chicken microRNAs. Our results have confirmed the expression of many microRNAs identified by sequence similarity and identified a pool of candidate novel microRNAs.
By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and ...IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-gamma synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.
The application of microarray technology to functional genomic analysis in the chicken has been limited by the lack of arrays containing large numbers of genes.
We have produced cDNA arrays using ...chicken EST collections generated by BBSRC, University of Delaware and the Fred Hutchinson Cancer Research Center. From a total of 363,838 chicken ESTs representing 24 different adult or embryonic tissues, a set of 11,447 non-redundant ESTs were selected and added to an existing collection of clones (4,162) from immune tissues and a chicken bursal cell line (DT40). Quality control analysis indicates there are 13,007 useable features on the array, including 160 control spots. The array provides broad coverage of mRNAs expressed in many tissues; in addition, clones with expression unique to various tissues can be detected.
A chicken multi-tissue cDNA microarray with 13,007 features is now available to academic researchers from http://genomics@fhcrc.org. Sequence information for all features on the array is in GenBank, and clones can be readily obtained. Targeted users include researchers in comparative and developmental biology, immunology, vaccine and agricultural technology. These arrays will be an important resource for the entire research community using the chicken as a model.
We present an analysis of the chicken (Gallus gallus) transcriptome based on the full insert sequences for 19,626 cDNAs, combined with 485,337 EST sequences. The cDNA data set has been functionally ...annotated and describes a minimum of 11,929 chicken coding genes, including the sequence for 2260 full-length cDNAs together with a collection of noncoding (nc) cDNAs that have been stringently filtered to remove untranslated regions of coding mRNAs. The combined collection of cDNAs and ESTs describe 62,546 clustered transcripts and provide transcriptional evidence for a total of 18,989 chicken genes, including 88% of the annotated Ensembl gene set. Analysis of the ncRNAs reveals a set that is highly conserved in chickens and mammals, including sequences for 14 pri-miRNAs encoding 23 different miRNAs. The data sets described here provide a transcriptome toolkit linked to physical clones for bioinformaticians and experimental biologists who wish to use chicken systems as a low-cost, accessible alternative to mammals for the analysis of vertebrate development, immunology, and cell biology.
The transcriptional effects of deregulated myc gene overexpression are implicated in tumorigenesis in a spectrum of experimental and naturally occurring neoplasms. In follicles of the chicken bursa ...of Fabricius, myc induction of B-cell neoplasia requires a target cell population present during early bursal development and progresses through preneoplastic transformed follicles to metastatic lymphomas. We developed a chicken immune system cDNA microarray to analyze broad changes in gene expression that occur during normal embryonic B-cell development and during myc-induced neoplastic transformation in the bursa. The number of mRNAs showing at least 3-fold change was greater during myc-induced lymphomagenesis than during normal development, and hierarchical cluster analysis of expression patterns revealed that levels of several hundred mRNAs varied in concert with levels of myc overexpression. A set of 41 mRNAs were most consistently elevated in myc-overexpressing preneoplastic and neoplastic cells, most involved in processes thought to be subject to regulation by Myc. The mRNAs for another cluster of genes were overexpressed in neoplasia independent of myc expression level, including a small subset with the expression signature of embryonic bursal lymphocytes. Overexpression of myc, and some of the genes overexpressed with myc, may be important for generation of preneoplastic transformed follicles. However, expression profiles of late metastatic tumors showed a large variation in concert with myc expression levels, and some showed minimal myc overexpression. Therefore, high-level myc overexpression may be more important in the early induction of these lymphomas than in maintenance of late-stage metastases.
MicroRNAs are short RNAs (~22 nt) expressed by plants, animals and viruses that regulate gene expression post-transcriptionally, and their importance is highlighted by distinct patterns of expression ...in many physiological processes, including development, hematopoeisis, stress resistance, and disease. Our group has characterized the microRNAs encoded by the avian herpesviruses; namely, oncogenic Marek's disease (MD) virus (MDV1), non-oncogenic MDV (MDV2) herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILTV).
MicroRNAs encoded by the avian herpesviruses were identified using next generation sequencing technologies (454, Illumina).
The microRNAs of each the avian herpesviruses have unique sequences, but the genomic locations are similar, in that the microRNAs tend to be clustered in the rapidly evolving repeat regions of the viral genomes. For a given viral species the microRNA sequence is highly conserved in different strains with the exception of a virulence-associated polymorphism in the putative promoter of the MDV1 microRNAs upstream of the meq oncogene. These microRNAs are relatively highly expressed in tumors produced by very virulent MDV1 isolates compared to tumors produced by less virulent strains. MDV1 and HVT encode homologs of the host microRNA, miR-221, which targets a gene important in cell cycle regulation. MDV1 encodes a microRNA (mdv1-miR-M4) that shares a seed sequence with miR-155, a microRNA important in immune function. Mdv-miR-M4 is highly expressed in MDV induced tumors, while miR-155 is present at very low levels.
MicroRNAs are highly conserved among different field strains of MDV1, and they are expressed in lytic and latent infections and in MDV1-derived tumors. This suggests that these small molecules are very important to the virus, and roles in immune evasion, anti-apoptosis, or proliferation are likely.
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